Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
  • C07H13/06—Fatty acids

Definitions

• the present invention relates to a novel method for producing N-acetylsilactosamin oligosaccharide, and more particularly, to a method for producing N-acetylsilactosamin oligosaccharide from keratan sulfate having adjusted sulfate group content by an enzyme reaction and a chemical reaction.
• the present invention also relates to a novel method for producing sulfated N-acetyllactosamine oligosaccharide.
• the present invention also relates to a novel N-acetyl-lactosamine oligosaccharide.
• Oligosaccharides and derivatives thereof having a N-acetylsyllactosamine (LacNAc) structure as a skeleton play important physiological functions in vivo as sugar chains capable of binding to selectin families. It has been revealed that the expression of these sugar chains on the cell surface is involved in specific adhesion between heterologous cells, and functional activators and inhibitors using these sugar chains are expected to be useful drugs Have been. In other words, its central compound, sialyl Lewis X CSLe ') and its related compounds block selectin molecules and inhibit adhesion of lymphocytes to endothelial cells, thereby reducing various diseases involving inflammation. It is thought to be helpful.
• Oligosaccharides and their derivatives having an N-acetyl lactosamine structure as a skeleton are expected to play an important role in the development of new drugs and in studies on biological activity in vivo. If a long-chain N-acetyllactosaminooligosaccharide, which is similar to the natural ligand sugar chain of the selectin family, is obtained as its basic skeleton, N-acetyllactosaminooligosaccharide, the N-acetyllactosaminooligosaccharide can be obtained.
• sialic acid residues, fucose residues, sulfate groups, ceramides, etc. to sugars as appropriate, it is possible to synthesize selectin family ligand sugar chains that are present only in trace amounts in nature. It is thought that it can be achieved.
• N-acetyl-lactosamine oligosaccharides especially long-chain N-acetyl-lactosamine oligosaccharides.
• Oligosaccharides having an N-acetyl lactosamine structure as a skeleton are naturally present in only trace amounts, and the synthetic production of N-acetyl lactosamine oligosaccharides in a chain is extremely difficult.
• a method for producing inexpensive N-acetyllactosamine oligosaccharide is desired.
• sulfated N-acetyl-lactosamine oligosaccharides are useful as intermediates for synthesizing N-acetyl-lactosamine oligosaccharides, especially long-chain N-acetyl-lactosamine oligosaccharides.
• this production method it is expected that it will lead to a stable supply of N-acetylsilactosaminoligosaccharide. Disclosure of the invention
• the present invention has been made in view of the above, and has a novel N-acetylacetyltosaminooligosaccharide, a novel method for producing an N-acetylethyltosaminooligosaccharide, which is a material for synthesizing a selectin family ligand sugar chain, Another object of the present invention is to provide a novel method for producing a sulfated N-acetyl lactosamine oligosaccharide.
• the present inventors have conducted intensive studies to solve the above problems, and as a result, adjusted the sulfate group content of keratan sulfate having an N-acetyl lactosamine structure as a basic skeleton, and obtained a keratan sulfate having an adjusted sulfate group content.
• the present inventors have found that N_acetyl lactosamine oligosaccharide (particularly, long-chain N-acetyl lactosamine oligosaccharide) can be obtained by decomposing to sulfated N-acetyl lactosamine oligosaccharide by an enzyme and then completely desulfating it, leading to the present invention.
• N_acetyl lactosamine oligosaccharide particularly, long-chain N-acetyl lactosamine oligosaccharide
• sulfated N-acetyl lactosamine oligosaccharide (especially long-chain sulfated N-acetyl lactosamine oligosaccharide) is obtained by adjusting the sulfate group content of keratan sulfate and enzymatically digesting the keratan sulfate having the adjusted sulfate group content. And found that the present invention was achieved.
• the present invention relates to an enzyme having an action of adjusting the sulfate group content of keratan sulfate and having an action of cleaving the glycosidic bond of keratan sulfate with the adjusted sulfate group content.
• an enzyme having an action of adjusting the sulfate group content of keratan sulfate and having an action of cleaving the glycosidic bond of keratan sulfate with the adjusted sulfate group content.
• Oligosaccharide production method also referred to herein simply as “Method 1 of the present invention”
• novel N-acetylacetyltosaminooligosaccharide also referred to herein simply as “oligosaccharide of the present invention”
• oligosaccharide of the present invention novel N-acetylacetyltosaminooligosaccharide
• the present invention provides a method for adjusting the sulfate group content of keratan sulfate, wherein the enzyme specificity of three types of keratan sulfate degrading enzymes having different substrate specificities depending on the presence or absence of a sulfate group in keratan sulfate having an adjusted sulfate group content.
• a method for producing a sulfated N-acetylacetyltosaminooligosaccharide (which may be simply referred to as "method 2 of the present invention” in the present specification), which comprises at least a step of reacting one or more enzymes belonging to any of them.
• the method 1 of the present invention and the method 2 of the present invention may be simply referred to as “the method of the present invention”.
• N-acetylsilactosamine refers to a structure (Gal GlcNAc) in which a galactose residue (Ga) and an N-acetylglucosamine residue (GlcNAc) are glycosidically bonded.
• Ga galactose residue
• GlcNAc N-acetylglucosamine residue
• N-acetylacetyltosamine includes a structure in which an N-acetylglycosamine residue (GlcNAc) and a galactose residue (Gal) are glycoside-linked (GlcNAc- Gal).
• N-acetyl lactosamine oligosaccharide refers to an oligosaccharide having at least one N-acetyl lactosamine structure, such as N-acetyl lactosamine itself or N-acetyl lactosamine via a glycosidic bond. Oligosaccharides consisting of repetition of trisamine are included. A sialic acid residue may be added to the non-reducing terminal. The non-reducing end and the reducing end may be any of a galactose residue and an N-acetylglucosamine residue. These are all referred to in this specification.
• N-acetylsacyl oligosaccharide That is, the oligosaccharides represented by the following general formulas (1) to (6) are all included in the “N-acetylsacyllactam oligosaccharide” of the present specification. (GlcNAc-Gal) n (1)
• Gal is a galactose residue
• Glc is an N-acetylglucosamine residue
• SA is a sialic acid residue,-is a glycosidic bond
• n is an integer from 1 to 6
• m Represents an integer from 1 to ⁇
• sulfated N-acetyl lactosamino oligosaccharide refers to an N-acetyl lactosamino oligosaccharide having a sulfate group.
• sulfated oligosaccharide means an oligosaccharide having a sulfate group.
• “to adjust the sulfate group content” means to increase (ie, to sulfate) or to decrease (ie, partially desulfurize) the sulfate group content so as to obtain a desired sulfate group content. Oxidizing) and without increasing or decreasing the sulphate content, but also at the desired sulphate content, without altering the sulphate content.
• partial desulfation means partial removal of a sulfate group, and differs from “complete desulfation” in that a part of the sulfate group remains.
• complete desulfation means removing substantially all of the sulfate groups, and is different from partially removing the sulfate groups.
• FIG. 1 is a graph showing the relationship between the time for partial desulfation of keratan sulfate by the methanol / hydrochloric acid method and the production rate of sulfated N-acetyllactosamine oligosaccharide of 6 to 12 saccharides.
• FIG. 2 is a graph showing the relationship between the temperature of the partial desulfation of keratan sulfate by the DMSO method and the rate of production of sulfated N-acetyl lactosamino oligosaccharides of 6 to 12 sugars.
• Figure 3 shows keratan sulfate partially desulfated by the DMS ⁇ method at various temperatures.
• Fig. 3 shows an elution curve obtained by subjecting a product obtained by the action of endo / 9-galactosidase or kerata 11- 11 derived from Escherichia freundi i to gel filtration.
• a sulfated N-acetyllactosamine oligosaccharide is obtained by adjusting the sulfate group content of keratan sulfate, and allowing the keratan sulfate degrading enzyme to act on the keratan sulfate having the adjusted sulfate group content.
• This is a method for producing N-acetylsilactosamin oligosaccharide, which comprises a step of completely desulfating sulfated N-acetyl lactosamine oligosaccharide.
• the method 2 of the present invention comprises adjusting the sulfate group content of keratan sulfate, and adjusting the sulfate specificity of the keratan sulfate to three types of keratan sulfate degrading enzymes having different substrate specificities depending on the presence or absence of a sulfate group.
• a method for producing a sulfated N-acetyllactosamine oligosaccharide comprising at least a step of acting one or more enzymes belonging to a group of enzymes.
• Keratan sulfate used in the method of the present invention
• Keratan sulfate has a repeating disaccharide (N-acetyllactosamine; LacNAc) in which a galactose residue (Gai) and an N-acetylglucosamine residue (GlcNAc) are glycosidically bonded. It is a glycosaminoglycan having 1 to 2 mol of sulfate groups per mol of the disaccharide.
• the sulfate group content of keratan sulfate varies depending on the animal species and organs, it is usually produced from raw materials such as cartilage fish such as sharks, cartilage of mammals such as whales and red sea lions, bone and cornea.
• the keratan sulfate used in the method of the present invention is not particularly limited, as long as it is generally available.
• the highly sulfated keratan sulfate (constituting disaccharide) in which a galactose residue which is a carbohydrate constituting sugar is sulfated is not particularly limited.
• Highly sulfated keratan sulfate containing 1.5 to 2 moles of sulfate groups per mole is sometimes referred to as keratan polysulfate.
• the galactose remaining The position of the sulfate group in the group is preferably the 6-position.
• Such highly sulfated keratan sulfate can be obtained, for example, from cartilage proteoglycans of cartilaginous fish such as sharks. Commercially available ones can also be used.
• the term “keratan sulfate” also includes highly sulfated keratan sulfate / keratan polysulfate unless otherwise specified.
• Methods for adjusting the sulfate group content of keratan sulfate include sulfation to a desired sulfate group content, partial desulfation, and desired sulfate group content without increasing or decreasing the sulfate group content. If it is, it also includes not changing the sulfate group content. Which sulfate group content adjustment method is selected depends on the sulfate group content of keratan sulfate used in the method of the present invention, the type of keratan sulfate degrading enzyme used in the method of the present invention, and the size of the target oligosaccharide. Thus, those skilled in the art can appropriately select.
• the sulfate group content is adjusted so that an oligosaccharide of a target molecular size can be obtained by the action of keratan sulfate degrading enzyme. More specifically, the average is 0.3 per mol of the constituent disaccharide. Adjust to the extent that ⁇ 1.5 moles of sulfate groups are present.
• the ratio of 1 1 or (2) described below is used as the keratan sulfate degrading enzyme. It is preferable to use keratan sulfate whose sulfate group content is adjusted to the extent that up to 1.5 mol of sulfate groups are present.
• an enzyme belonging to the enzyme group (2) or (3) described below is used as the keratan sulfate-decomposing enzyme, the amount of the sulfate group in the range of 0.3 to 0.8 mol per mol of the constituent disaccharide is considered. It is preferable to use keratan sulfate whose sulfate group content is adjusted.
• the partial desulfation can be carried out by a known desulfation method for glycosaminoglycin, the temperature during the desulfation reaction, the time for the desulfation reaction, and the amount of the desulfating agent so that substantially all of the sulfate groups are not removed. It is necessary to set reaction conditions such as concentration. That is, the temperature during the desulfation reaction, the time for the desulfation reaction, the concentration of the desulfating agent, etc. are set lower than the reaction conditions under which substantially all of the sulfate groups are removed.
• reaction conditions are: --Keratan sulfate used, type of keratan sulfate degrading enzyme used for cleavage of glycoside bond of keratan sulfate with adjusted sulfate group content, molecular size of target N-acetylacetylsamin oligosaccharide (molecular weight, number of sugar chains) Those skilled in the art can appropriately set the values according to the above.
• Known methods for desulfating glycosaminoglycin include, for example, acid hydrolysis, alkali decomposition, and heating in an organic solvent (Method in Carbohydrate Chemist II, vol. VI II, p281-). 289, 1980). That is, acids, alkalis, organic solvents and the like can be used as desulfating agents.
• an inorganic acid, an organic acid, a strongly acidic cation exchange resin, particularly a sulfonic acid resin or the like can be used as an acid, and water, methanol, or the like can be used as a reaction solvent. it can.
• an anhydrous methanol solution containing acetyl chloride it is preferable to use an anhydrous methanol solution containing acetyl chloride.
• a non-protonic solvent such as dimethyl sulfoxide (DMS 0; N, N-dimethylformamide (DMF), pyridine) can be used as the organic solvent.
• DMS 0 dimethyl sulfoxide
• DMF N-dimethylformamide
• pyridine a non-protonic solvent
• a method of heating pyridinum keratan sulfate in water or a DMSO solution containing 5 to 10% methanol at a temperature in the range of 20 to 100 ° C is widely used (Shinsei Kagaku Kenkyusho 3, Carbohydrate II, (Tokyo Kagaku Dojin, p325-326, 1989), and it is preferable to use this method also in the method of the present invention.
• a desulfation method using a silylating agent can also be used.
• Japanese Patent Application Laid-Open No. 5-230900 discloses that an organic base salt of a sulfated saccharide is reacted with N.0-bis (trimethylsilyl) acetamide in the presence of the organic base. Accordingly, a method for producing a desulfated saccharide characterized by selectively desulfating a sulfate group bonded to a primary hydroxyl group of a sulfated saccharide has been disclosed. it can.
• Organic base salts of keratan sulfate include aromatic amines such as pyridine, dimethylaniline and getylaniline; trimethylamine, triethylamine, tributylamine, ⁇ , ⁇ -diisopropylethylamine, trioctylamine,. ⁇ .
• trimethylsilylation reagents N, 0bis (trimethylsilyl) acetamide (BTSA), N, 0bis (trimethylsilyl) trifluorase amide (BTSFA), N, 0 bis (trimethylsilinole) difluoroacetamide, N, 0 bis (trimethylsilyl.) Monofluoroacetamide, etc. are added, and the reaction is allowed to proceed at room temperature to 100 ° C to achieve desulfation. It can be carried out.
• a process for producing a desulfated sugar which comprises subjecting to a desulfation reaction in the presence of a silylated ⁇ ⁇ ⁇ represented by the formula: This method can also be used. Also in this method, it is preferable to react the silylated ⁇ ⁇ ⁇ represented by the above general formula (A) with the organic base salt of the sulfated saccharide, and as the organic base salt, the above-mentioned JP-A-5-230900 is used. The same organic base salts as those described in the gazette can be used.
• (R Si O is, for example, trimethylsilyloxy, triethylsilyloxy, dimethylisopropyl virsilyloxy, isopropyldimethylsilyloxy, methyl-dibutylbutylsilyloxy, t-butyldimethylsilyloxy
• Examples of the silylating agent represented by the above general formula (A) include 2-trimethyl butyl diphenylxylsilyloxy, triisopropylsilyloxy, etc. Desulfation can be performed by reacting at a reaction temperature between room temperature and 100 ° C.
• R 1 is the same or different and represents a hydrogen atom or a halogen atom
• R 2 represents a lower alkyl group
• R 3 represents the same or different and represents a lower alkyl group, an aryl group or a halogen atom.
• R 1 in the above formula (B) is the same or different and represents a hydrogen atom or a halogen atom such as fluorine
• R 2 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl. , Tert-butyl, pentyl, isopentyl, hexyl and the like, and represents a lower alkyl group having 1 to 6 carbon atoms
• R 3 is the same or different, and is the same as the above, such as a lower alkyl group, an aryl group such as phenyl, chlorine, fluorine, etc.
• (R 3 ) 3 Si in the above general formula (B) includes trimethylsilyl, triethylsilyl, dimethylisopropylsilyl, isopropyldimethylsilyl, methyldi-tert-butylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisobromide
• a bilsilyl group is exemplified.
• the most preferred silylating agent represented by the above general formula (B) is N-methyl-N-trimethylsilyl acetate (MTMSA) or N-methyl-N-trimethylsilyl trifluoroacetate.
• MTSTFA 1 can be used. Desulfation can be performed by reacting at room temperature to 100. Further, an enzyme having an action of desulfating keratan sulfate may be used. These glycosaminoglycans may be used. By appropriately setting the reaction conditions in the known desulfation method described in (2), keratan sulfate having an appropriately reduced number of sulfate groups can be obtained.
• Sulfation can be performed by a known sulfation method for glycosaminoglycin, and is not particularly limited. Examples thereof include a method described in JP-A-61-47071. Is mentioned. As described above, since keratan sulfate generally has a large sulfate group content, adjustment of the sulfate group content of keratan sulfate is most often performed by partial desulfation. However, if keratan sulfate having an extremely low sulfate group content is available and can be used, the sulfate group content can be adjusted by sulfation.
• the sulfate group content is adjusted by sulfation, it is preferable that only the sixth position of the galactose residue and / or the N-acetyl dalcosamine residue be sulfated. It is preferable to select a specific sulfation method.
• galactose residues of keratan sulfate and / or N-acetylglucosa An enzyme (sulfotransferase) that specifically transfers a sulfate group to position 6 of the min residue may be used.
• the sulfate group content of keratan sulfate or the sulfate group whose sulfate group content has been adjusted can be quantified by a known method, and can be evaluated based on, for example, the results obtained by the action of keratan sulfate degrading enzyme. .
• the evaluation may be based on the number of moles of the sulfuric acid group per mole of the constituent disaccharide.
• the number of moles of sulfate groups per mole of the constituent disaccharide can be determined by determining the number of sugar residues by quantifying galactose-N-acetylglucosamine by an anceron method and amino sugar analysis.
• the sulfate group content can be determined by ion chromatography analysis of the hydrochloric acid decomposed product, and the content can be determined by calculating from these values. If the sulfate group content of the keratan sulfate as the raw material is the desired sulfate group content, it is not necessary to actively adjust the sulfate group content (that is, sulfation or partial desulfation), and the present invention is directly used. Can be used.
• keratan sulfate generally has a large sulfate group content, and it is preferable to use highly sulfated keratan sulfate / keratan polysulfate as a raw material. “Adjustment of the sulfate group content” is often and preferably performed by partial desulfation. Further, it is preferable to select a keratan sulfate degrading enzyme (to be described later) to be used accordingly.
• the keratan sulfate degrading enzyme that can be used in the method of the present invention is not particularly limited as long as it has an action of cleaving the glycosidic bond of keratan sulfate, and may be any of the following enzyme groups (1) to (3). It is preferable to use one or more enzymes belonging to the group.
• Enzymes (1) Enzymes having the following action and substrate specificity
• the enzyme group (1) is preferably endo-galactosidase.
• Enzymes included in the enzyme group (1) include, for example, an ent / 3 / 3-galactosidase derived from Escherichia freundii (H. Nakagawa, T. Yamada, J L. Chien, A. Gardas) , M. Kitamikado, S C. Li, Y-i. Li, J. Biol. Chem., 255, 5955 (1980); in this specification, it may be simply referred to as “GalaseJ”. .
• Enzymes (2) Enzymes with the following action and substrate specificity
• the galactose residue involved in the galactose residue has a sulfate group at position 6 and does not act on the —galactoside bond.
• Enzyme group (2) is preferably endo; S-galactosidase.
• Enzymes included in the enzyme group (2) include, for example, Endo-S—galactosidase derived from Pseudomonas sp. IFO-13309 (K. Nakazawa, N. Suzuki, S. Suzuki, J. Biol. Chem., 250, 905 (1975); K. Nakazawa, S. Suzuki, J. Biol. Chem., 250, 912 (1975); and Japanese Patent Publication No. 57-4121336 Endos produced by Pseudomonas reptilivora — yS —galactosidase (these are sometimes referred to simply as “Kerata eleven” or “KSase”) ) Is preferred.
• Endo-S galactosidase derived from Pseudomonas sp. IFO-13309
• Enzymes (3) Enzymes having the following action and substrate specificity (a) Action:
• N-Acetylglucosamine Residue involved in N-acetylglucosamine bond has a sulfate group at the 6-position and is adjacent to the non-reducing terminal side of the N-acetylglucosamine residue It does not have a sulfate group at position 6 of the galactose residue and acts on the 3-N-acetyldarcosamide bond.
• Enzyme group (3) is preferably L / 3-N-acetylglucosaminidase.
• the enzymes included in the enzyme group (3) include, for example, those derived from Bacillus sp. Ks36 (Bacillus Ks36) (Shinichi Hashimoto, Kiyoshi Morikawa, Hiroshi Kikuchi, Keiichi Yoshida, Kiyochika Tokuyasu, Biochemistry, 60 , 935 (1988); herein, it may be simply referred to as “keratanase II” or “KSaseI I”), or derived from Bacillus circulans KsT202 (Bacillus circulans KsT202) (WO96). / Described in US Pat. No.
• Enzymes (1) and (2) are both endo-yS-galactosidase enzymes, and their substrate specificities differ depending on the sulfate groups present in the sugar chains.
• the enzyme group (3) is an end- ⁇ g-N-acetylglucosaminidase type enzyme.
• the reducing end of the oligosaccharide obtained by enzymatic digestion is a galactose residue (Gal) when an endo-galactosidase type enzyme is used, and the endo- / 3-N-acetyl When a glucosaminidase type enzyme is used, it is an N-acetyl glucosamine residue (GicNAc).
• Gal indicates a galactose residue
• GlcNAc indicates an N-acetylglucosamine residue
• (6S) indicates that the hydroxyl group at position 6 is sulfated.- and-indicate a glycosidic bond.
• Keratan sulfate derived from cartilage has a high content of disulfated N-acetyl lactosamine (LacNAc-diS) residues, and particularly high content of shark-derived keratan sulfate. Therefore, for such keratan sulfate, the enzymes of the enzyme groups (1) and (2) (E-Galase and KSase, for example) hardly act, but on the contrary, they are almost completely degraded by enzymes of the enzyme group (3) (for example, KSase II), and are easily converted into disaccharides and tetrasaccharides.
• the enzymes of the enzyme groups (1) and (2) E-Galase and KSase, for example
• enzymes of the enzyme group (3) for example, KSase II
• the molecular size of the N-acetyllactosamine oligosaccharide finally obtained by the method of the present invention can be controlled by a combination of the above substrate specificity of the enzyme and the degree of partial desulfation of keratan sulfate. That is, depending on the specificity of the enzyme used, the molecular size can be reduced by adjusting the degree of partial sulfation to increase the action of the enzyme. Conversely, adjusting the degree of partial sulfation to reduce the action of the enzyme increases the molecular size.
• the keratan sulfate having a sulfate group-containing IS adjusted and one or more enzymes belonging to any of the above enzyme groups (1) to (3) are allowed to coexist, If a large amount of the enzyme is to be treated, an immobilized enzyme in which the enzyme is bound to an appropriate solid-phase bead, etc., or an ultrafiltration membrane, a dialysis membrane, etc. It is preferable to make the enzyme act continuously using a reactor or the like.
• sulfated N-acetylacetylsamin oligosaccharide having a desired molecular size can be obtained.
• the sulfated N-acetylsuctosaminooligosaccharide produced by the enzyme treatment be fractionated according to the molecular size of the oligosaccharide.
• the method of fractionation according to the molecular size is not particularly limited, and examples thereof include gel filtration chromatography using various gel filtration carriers and a fractionation method using an organic solvent.
• the gel filtration carrier is not particularly limited as long as it is a carrier made for the purpose of a molecular sieve, and examples thereof include crosslinked acrylamide, dextran, agarose, and crosslinked particles of a polysaccharide such as cellulose.
• a carrier having a degree of cross-linking suitable for each oligosaccharide can be appropriately selected according to the molecular size of the target oligosaccharide.
• oligosaccharides up to about 8 octasaccharides can be separated according to the molecular size of each oligosaccharide, but the separation ability of oligosaccharides longer than that It decreases as the size increases. In this case, it is fractionated as a mixture of oligosaccharides of several molecular sizes.
• the oligosaccharide mixture may be fractionated in advance by anion exchange resin according to the sulfate group density, whereby the separation ability by gel filtration chromatography can be improved.
• anion exchange resin generally, when the density of sulfate groups is the same, the larger molecular size is strongly adsorbed on the anion exchange resin. Therefore, mutual separation is possible.
• the sugar chain skeleton of the sulfated N-acetyl lactosamine oligosaccharide thus obtained is represented by the following general formulas (1) to (6).
• the following general formulas (1) and (2) belong to the enzyme groups (1) and Z or the enzyme group (2) in keratan sulfate having a sulfate group content adjusted in the method of the present invention. It is a backbone of sulfated N-acetyllactosamine oligosaccharide that can be obtained by the action of an enzyme.
• the following general formulas (3) and (4) represent sulfated N-acetyl lactides obtainable when an enzyme belonging to the enzyme group (3) is allowed to act on keratan sulfate having an adjusted sulfate group content in the method of the present invention. It is the skeleton of tosamino glycol.
• the following general formulas (5) and (6) represent the enzymes belonging to the enzyme group (1) and / or (2) in the keratan sulfate whose sulfate group content has been adjusted in the method of the present invention, and the enzyme group (3 ) Is a skeleton of sulfated N-acetyllactosamine oligosaccharide which can be obtained when both enzymes are acted on.
• Gai is a galactose residue
• GlcNAc is an N-acetylglucosamine residue
• SA is a sialic acid residue,-is a glycosidic bond
• n is an integer of 1 to 6
• ⁇ is Represents an integer from 1 to 10)
• keratanase II which is an enzyme belonging to the enzyme group (3) is allowed to act on keratan sulfate having an adjusted sulfate group content, for example, a sulfate having a skeleton represented by the following formula: ⁇ -Acetyl lactosamin oligosaccharide is obtained.
• the sulfated N-acetyllactosamine oligosaccharide having the skeleton represented by the above formulas (7) to (14) is used only for the sulfated N-acetyllactosamine oligosaccharide having the skeleton represented by the general formulas (3) and (4). — It is only a specific example of acetyl lactosamine oligosaccharide and is not limited thereto.
• N-acetyl lactosamino oligosaccharide having a skeleton represented by any of the above general formulas (1;) to (6) by a method described below an N-acetyl laclac represented by the following general formula is obtained.
• Tosamine oligosaccharides can be obtained.
• GlciMAc N-acetylglycosamine residue
• SA sialic acid residue
• n is an integer of 1 to 6
• m Represents an integer of 1 to 10, respectively.
• a sulfated N-acetyl lactosamine oligosaccharide having a skeleton represented by the above formulas (7) to (14) is completely desulfated by a method described below, and is then represented by the following formula. Can be obtained.
• Gal-GlcNAc-Gal-Glc Ac-Gal GlcNAc (15) Gal Glc Ac-Gal -GlcNAc Gal-GlcNAc-Gal -GlcNAc (16) Gal-GlcNAc Gal ⁇ GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal -GlcNAc ( 17) Gal GlcNAc Ga GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gai GlcNAc Gal -GlcNAc (18) SA-Gal Glc Ac-GaJ -GlcNAc Gal-GlcNAc (19) SA Gal-GlcNAc-Gal-Gl cNAc -Gal Gl cNAc -Gal -GlcNAc (20) SA-Gal -GlcNAc -Gal GlcNAc Gal-GlcNAc Gal-Gl
• N-acetyl lactosamin oligosaccharides represented by the above formulas (15) to (22) are only sulfated N-acetyl lactates having a skeleton represented by the above general formulas (3) and (4). This is only a specific example of the N-acetyl lactosamine oligosaccharide obtained from the samin oligosaccharide, but is not limited thereto.
• n is an integer of 1 to 6, and a long-chain N-acetyl lactosamine oligosaccharide, for example, an n-type integer of 3 to 6 is preferable, and n is 5 to 6. Those which are integers of 6 are particularly preferred.
• m is an integer of 1 to 10 and a long-chain N-acetyllactosamine oligosaccharide, for example, m is preferably an integer of 3 to 10; Those which are integers of 5 to 10 are particularly preferred.
• a preferred sialic acid residue (SA) is N-acetylneuraminic acid residue ⁇ (NeuAc).
• the glycosidic bond between the sialic acid residue (SA) and the galactose residue (Gal) adjacent to the reducing end of the sialic acid residue (SA) is a 1,2,3 glycosidic bond. Or, it is preferably a 1,2,6-glycosidic bond.
• glycosidic bond between the galactose residue (Gal) and the N-acetylacetylglucosamine residue (GicNAc) adjacent to the reducing end of the galactose residue (Gal) is represented by the following formula: , 4 glycosidic bonds are preferred.
• the glycosidic bond between the N-acetylglycosamine residue (GlcNAc) and the galactose residue (Gai) adjacent to the reducing end side of the N-acetylglycosamine residue (GlcNAc) is , 3-1, 3 glycosidic bonds are preferred.
• the N-acetyl-lactosamine oligosaccharide having a sialic acid residue and the sulfated N-acetyl-lactosamine oligosaccharide have a sialic acid residue.
• the desulfation conditions described above that is, the conditions for complete desulfation while retaining sialic acid residues
• the invention also includes an invention relating to a method for completely desulfating an acetylsilactamsine oligosaccharide while retaining a sialic acid residue.
• the method for completely desulfating a sulfated N-acetyl lactosamine oligosaccharide includes, for example, acid hydrolysis, hydrolysis, and heating in an organic solvent in the same manner as the aforementioned partial desulfation of keratan sulfate.
• Examples thereof include a method using a silylating agent and a method using an enzyme, but the reaction conditions are different from those for partial desulfation.
• the dimethylsulfoxide method as a desulfating method using an organic solvent, the methanolic hydrochloric acid method as a desulfating method by acid hydrolysis, and the trisulfating method as a desulfating method using a silylating agent are described below. The method for complete desulfation is described in more detail below.
• Sulfated N-acetyllactosamine Oligosaccharide is dissolved in water and passed through a strong thione-exchange resin (H-type) to remove free sulfate groups, and then neutralized with pyridine to give sulfated N-acetyllactide. It is a pyridinium salt of tosamine oligosaccharide.
• Hydrogen chloride gas is passed through a dry solvent from which water has been sufficiently removed to form a methanol-hydrochloric acid solution.
• the solution is diluted with dry methanol and adjusted to 0.01 to 1N, preferably to 0.1N.
• Complete desulfation can be achieved by dispersing well-dried sulfated N-acetyllactosaminooligosaccharide in methanolic hydrochloric acid solution and stirring for 0.5 to 50 hours at 4 to 30 hours, preferably for 15 hours at room temperature. it can.
• N-acetyl lactosamine oligosaccharide is dissolved in a pyridine solution, and BTSA is added at a 5- to 16-fold molar amount, preferably 10-fold molar amount, based on the sulfate group, and the BTSA is added at a concentration of 0.5 to 90 at 40 to 90 ° C.
• Complete desulfation can be achieved by reacting for 3 hours, preferably for 1 hour at 80 ° C.
• the desulfation rate by this method has been confirmed by the inventors to be arbitrarily adjustable by changing the temperature and the treatment time.
• the method of complete desulfation is not limited to these.
• N-acetyl lactosamino oligosaccharide By completely desulfating the sulfated N-acetyl lactosamino oligosaccharide, N-acetyl lactosamino oligosaccharide can be obtained.
• N-acetyl lactosamine oligosaccharide For the fractionation of N-acetyl lactosamine oligosaccharide, the same method for separation and purification of sugar chains can be used as in the above-described method for sulphated N-acetyl lactosamine oligosaccharide.
• adsorption chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration, gel permeation chromatography, filter paper electrophoresis, filter paper chromatography, fractionation with organic solvents, or combinations thereof It can be performed by operation, but is not limited to these.
• the method for separating N-acetylsyllactosamine oligosaccharide having a sialic acid residue (sialyl N-acetylsyllactosamine oligosaccharide) from N-acetylsyllactosamine oligosaccharide is not particularly limited, either.
• ion-exchange chromatography This can be done by photography.
• the method of mutual separation of N-acetylsyllactosamine oligosaccharides having different molecular sizes is not particularly limited, but, for example, a methanol concentration of 0 to 20% is determined by chromatography using a reversed-phase column. It can be separated by raising it.
• the oligosaccharide of the present invention N-acetyl lactosamine oligosaccharide
• the oligosaccharide of the present invention will be described later.
• the method of the present invention preferably comprises adjusting the sulfate group content of keratan sulfate, and converting the keratan sulfate having the adjusted sulfate group content to one of the above enzyme groups (1) to (3) or
• the method includes a step of obtaining a sulfated N-acetyl lactosamine oligosaccharide by treatment with two or more enzymes (step 1), and a step of completely desulfating the sulfated N-acetyl lactosamine oligosaccharide (step 2).
• Step 1 preferably includes a step of fractionating sulfated N-acetylsyllactosaminooligosaccharide
• step 2 preferably includes a step of fractionating N-acetylsyllactosaminooligosaccharide.
• the method includes a step of fractionating acetyl lactosamine oligosaccharide
• step 1 includes a step of fractionating sulfated N-acetyl lactosamine oligosaccharide
• step 2 includes N-acetyl lactosamine oligosaccharide. It is particularly preferable to include a step of fractionating
• the conditions for partial desulfation or sulfation of keranic sulfate are conditions in which an enzyme capable of cleaving the glycosidic bond of keratan sulfate does not lose its activity.
• the partial desulfation or sulfation of keratan sulfate and the decomposition of keratan sulfate having an adjusted sulfate group content may be performed simultaneously (for example, in the same reactor).
• keratan sulfate which can be usually obtained is partially desulfated in advance, so that the above-mentioned enzyme does not act on keratan sulfate as it is.
• an oligosaccharide having a target molecular size can be obtained more efficiently.
• the non-reducing terminal sugar of the N-acetyl lactosamin oligosaccharide represented by the general formulas (1;) to (6) produced by the method of the present invention is added to the N-acetyl lactosamin oligosaccharide.
• an N-acetylsacralactosin oligosaccharide having a reduced number of sugar residues by one can be obtained. Specific examples are shown below.
• N_acetyl lactosaminooligosaccharides of the following general formulas (23) to (24) can be obtained.
• N-acetyl-lactosamine oligosaccharides of the following general formulas (25) to (26) can be obtained.
• Gal represents a galactose residue
• GlcNAc represents an N-acetylglycosamine residue
• ⁇ represents an integer of 2 to 6, respectively.
• N-acetyl neuraminidase for example, N-acetyl lactosamine oligosaccharide from which sialic acid has been removed can be obtained.
• the oligosaccharide of the present invention is an oligosaccharide obtained by the following method and having the following physicochemical properties.
• the sulfate group content of keratan sulfate is adjusted, and an enzyme having a function of cleaving the glycosidic bond of keratan sulfate is caused to act on the keratan sulfate having the adjusted sulfate group content to obtain a product, and then the product is completely desulfated.
• a method comprising:
• the galactose residue content measured by the anthrone method is 35 to 50% by weight.
• the molecular weight measured by mass spectrometry is 1000 to 220. Adjustment of the sulfate group content is preferably performed by partial desulfation.
• the above method preferably includes at least the following steps 1 and 2.
• Step 1 The sulfate group content of keratan sulfate is adjusted, and one or more enzymes belonging to any of the following enzyme groups (1) to (3) are produced from the keratan sulfate having the adjusted sulfate group content. To obtain a sulfated oligosaccharide.
• Enzymes (1) Enzymes having the following action and substrate specificity
• Enzymes (2) Enzymes with the following action and substrate specificity
• Enzymes (3) Enzymes having the following action and substrate specificity
• a sulfate group at the 6-position of the N-acetylglycosamine residue involved in N-acetylglycosaminide binding, and a galactose adjacent to the non-reducing terminal side of the N-acetylglycosamine residue It does not have a sulfate group at position 6 of one residue and acts on the / 3-N-acetylglucosaminide bond.
• 3-galactose residue adjacent to the 6th position of the N-acetylglycosamine residue involved in N-acetylacetylglucosaminide binding and the non-reducing terminal side of the N-acetylglucosamine residue Does not have a sulfate group at either of the 6-positions, does not act on the /? — N-acetylglucosaminide bond.
• Step 2 A step of completely desulfating a sulfated oligosaccharide.
• the enzyme contained in the enzyme group (1) is preferably an endo-galactosidase, for example, an endo-galactosidase derived from Escherichia freundii (scheridii ⁇ freundii). Nakaga a, T. Yatnada, J-shi Chien, A. Gardas, M. Kitamikado, S-C. Li, Y T. Li, J. Biol. Chem., 255, 5955 (1980); , Or simply “E-Galase”).
• endo-; 9-galactosidase is preferable, and for example, an end derived from Pseudomonas sp-IFO-13309 strain — S-galactosidase ( K. Nakazawa, N. Suzuki, S. Suzuki, J. Biol. Chem., 250, 905 (1975), K. Nakazawa, S. Suzuki, J. Biol. Chem., 250, 912 (1975)) Japanese Patent Publication No. 57-4121236 discloses an endo- / 3-galactosidase produced by Pseudmonas reptilivora (these are referred to simply as "keratana And “KSase”), and are preferred.
• the enzyme included in the enzyme group (3) is preferably endo-N-acetylglycosaminidase, for example, an endo-N-acetylglucosaminidase derived from Bacillus sp. S36. Ichize [Shinichi Hashimoto, Kiyoshi Morikawa, Hiroshi Kikuchi, Keiichi Yoshida, Kiyochika Tokuyasu, Biochemistry, 60, 935 (1988); In the present specification, only “Kerayuna II” or “KSaseII” And Bacillus sa —Endo / 9—N-acetyl-D-cosaminidase derived from Bacillus circulans KsT202 (described in WO 96/16166), and the like, are preferred.
• the oligosaccharide of the present invention more preferably has a sialic acid content of 10 to 20% by weight as measured by the thiobarbituric acid method in addition to the above-mentioned physicochemical properties. Further, the present invention provides a novel N-acetyl lactosamine oligosaccharide represented by any one of the following general formulas (1) to (6).
• Ga] is a galactose residue
• G] cNAc is an N-acetylglucosamine residue
• SA is a sialic acid residue
• n is an integer of 1 to 6.
• m represents an integer of 1 to 10.
• ⁇ is not 1.
• the method for producing the oligosaccharide of the present invention is not particularly limited as long as the oligosaccharide of the present invention can be obtained, but is preferably obtained by the method of the present invention.
• the method of the present invention has been described above.
• oligosaccharide of the present invention is produced by the method of the present invention, for example,!;
• a galactosidase type enzyme for example, an enzyme belonging to enzyme group (1) or (2) may be used.
• an enzyme of the end-3-N-acetylglucosaminidase type for example, belonging to the enzyme group (3) in the method of the present invention. Enzyme).
• an endo-type; S-galactosidase type enzyme for example, enzyme group (1) or ( Enzyme belonging to 2)
• endo-1 / 3-N-acetylglucosaminida enzymes of the enzyme type (for example, enzymes belonging to the enzyme group (3)) may be used.
• the sialic acid residue may be an N-acetyl neuraminic acid residue or an N-glycolyl neuraminic acid residue.
• N-acetyl neuraminic acid residue NeAc
• n is an integer of 1 to 6 (however, in the above formulas (1) and (3), n is not 1), but a long-chain N-acetyl lactosamino oligo is used.
• Sugars for example, those having an integer of 3 to 6 are preferred, and those having an integer of 5 to 6 are particularly preferred.
• ⁇ is an integer of 1 to 10; however, an N-acetylacetylsamin oligosaccharide having a chain length, for example, m is preferably an integer of 3 to 10 is preferable. Those in which m is an integer of 5 to 10 are particularly preferred.
• the glycosidic bond between the sialic acid residue (SA) and the galactose residue (Gai) adjacent to the reducing end of the sialic acid residue (SA) is an ⁇ -2,3 glycosidic bond or It is preferably an a-2, 6 glycosidic bond.
• the glycosidic bond between the galactose residue (Gal) and the N-acetylglucosamine residue (GlcNAc) adjacent to the reducing end of the galactose residue (Gal) is represented by / 3 — It is preferably a 1,4-glycosidic bond.
• glycosidic bond between the N-acetylglucosamine residue (GlcNAc) and the galactose residue (Gal) adjacent to the reducing end of the N-acetylglucosamine residue (GicNAc) is ⁇ -1 , 3 glycosidic bonds are preferred.
• the conditions for complete desulfation of sulfated ⁇ -acetylacetyltosamino-oligosaccharide having a sialic acid residue have been reported. This is the first condition found by the present inventors.
• the acetylacetyllactosamine oligosaccharide having a sialic acid residue are particularly preferable.
• the analysis of the sugar composition of the oligosaccharide of the present invention is carried out by a method usually used in the field of sugar chain engineering, for example, an anthrone method (quantification of galactose residues), an amino sugar analysis (p-acetylglycol). Quantification of cosamine residues) and thiobarbituric acid method (TBA method; quantification of sialic acid residues) are not limited to these. Further, the sulfate group content is not limited to this method, which can be analyzed by decomposing the oligosaccharide of the present invention with hydrochloric acid and analyzing it by ion chromatography.
• the method for analyzing the molecular size of the oligosaccharide of the present invention is not particularly limited, but it can be determined by, for example, gel filtration chromatography or analyzing the oligosaccharide after E-Galase treatment.
• the sugar chain sequence of the oligosaccharide of the present invention can be examined by, for example, a sequential decomposition method using glycosidase.
• the oligosaccharide of the present invention can be identified.
• the oligosaccharide of the present invention obtained by the method of the present invention can be used for confirmation.
• a sialic acid residue, a fucose residue, a sulfate group, a ceramide, and the like can be appropriately added to the oligosaccharide of the present invention.
• the addition of these substances is preferably carried out by using an enzyme which can also be carried out by a chemical method.
• the fucose residue when adding a fucose residue to the oligosaccharide of the present invention, the fucose residue may be transferred to the oligosaccharide of the present invention using fucosyltransferase.
• the fucose residue is preferably bound to the N-acetylglycosamine residue, and more preferably to the N-acetylglucosamine residue via a 1,3 glycoside bond. Therefore, it is preferable to use fucosyltransferase having such an action.
• the sialic acid residue is added to the oligosaccharide of the present invention represented by the above formula (1), (3), (5) or (6)
• the sialic acid residue is added to the oligosaccharide of the present invention using sialyltransferase. Should be transferred.
• the sialic acid residue be bound to the galactose residue, and more preferably that the non-reducing terminal galactose residue be bound by a ⁇ -2,3 glycosidic bond or an ⁇ -2,6 glycosidic bond.
• it is attached to a residue. Therefore, it is preferable to use a sialyltransferase having such an action.
• the sulfate group when added to the oligosaccharide of the present invention, the sulfate group may be transferred to the oligosaccharide of the present invention by using sulfotransferase.
• Sulfotransferases include chondroitin 6-sulfotransferase (Biol. Chem., 268 (29), 21968 21974, 1993).
• These enzymes can be appropriately selected and used according to the target oligosaccharide, may be used alone, may act on a plurality of enzymes simultaneously, or may act on a plurality of enzymes sequentially. You may. Example
• Keratan sulfate (hereinafter also referred to as K S) was prepared from shark cartilage according to the literature (Biochemistry, 33, 746-752, 1961). The number of moles of sulfate groups per mole of the constituent disaccharide was 1.7.
• FIG. 1 shows the relationship between the time for partial desulfation of KS by the methanol-hydrochloric acid method and the production rate of sulfated N-acetyl lactosamine oligosaccharide of 6 to 12 saccharides.
• the production rate of sulfated N-acetylsyllactosamine oligosaccharide of 6 to 12 sugars was calculated based on the elution curve of sulfated N-acetyllactosamine oligosaccharide obtained by gel filtration and the total area of the figure surrounded by the time axis. It was calculated as the ratio of the area occupied by the fraction corresponding to the sulfated N-acetyllactosamine oligosaccharide of 212 sugar.
• sulfation of 6- to 12-saccharides without partial desulfation of KS was obtained for N-acetyllactosaminooligosaccharides.
• the production rate was about 10%.
• partial desulfation of KS by the methanol / hydrochloric acid method was performed within 40 hours at 4, the yield of sulfated 6- to 12-saccharide N-acetylacetyltosaminooligosaccharides was about 2 to 4 Doubled.
• the production rate is about 30% or more, and when the partial desulfation time is 5 to 10 hours, the production rate is Reached about 40%. From this, when enzymatic digestion of KS with 1 U of KSase II at 37 ° C for 12 hours to obtain sulfated N-acetyl lactosamino oligosaccharide of 6 to 12 saccharides, KS is converted to 4 ° C by the methanol-hydrochloric acid method.
• the time for partial desulfation in C is preferably 40 hours or less (excluding 0 hours), more preferably 2.5 to 20 hours, and particularly preferably 5 to 10 hours. Will be understood.
• the freeze-dried product 800 mg of KS Pirijiniumu salt was dissolved in 80 ml of 90% DMSO (10% H 2 0), divided into 20 ml Dzu' 4 fractions, respectively 40, 50, 60, 70 or 80 ° C For 3 hours, and the reaction was stopped by adding an equal volume of water. After dialysis of each reaction solution in running water overnight, the solution was concentrated to 4 ml, divided into two equal portions of 2 ml each, and 250 mM acetate buffer (pH 6) containing 10 mM calcium acetate was added to one side. 0.5 ml each of 100 mM acetate buffer (pH 6.5) was added to the mixture.
• the former was Escherichia freundii end- ⁇ -galactosidase (E-Galase, manufactured by Seikagaku Corporation; 1 unit (II) was pH 5.8).
• E-Galase Escherichia freundii end- ⁇ -galactosidase
• 1 unit (II) was pH 5.8.
• the latter in the KSase II respectively added 1 Yuni' bets, at 37 Q C Enzymatic digestion was performed for 5 hours.
• Fig. 2 shows the relationship between the temperature during the partial desulfation of KS by the DMSO method and the rate of production of sulfated N-acetylacetylsosamin oligosaccharides of 6 to 12 sugars.
• the production rate of sulfated N-acetyl lactosamine oligosaccharide of 6 to 12 sugars was calculated in the same manner as in the above-mentioned 11-11. As shown in Fig.
• KSase II When KSase II is used in enzymatic digestion, when the temperature of the partial desulfation of KS by the DMSO method is about 65 ° C or higher, sulfation of 6- to 12-saccharide N-acetyl lactosamin oligosaccharide The production rate was about 20% or more, and reached 30% when the temperature of partial desulfation was 70, and reached about 40% when the temperature was 80 ° C. Based on these results, sulfated 6- to 12-saccharide N-acetyllactosaminooligosomes were obtained by enzymatic digestion of KS at 37 ° C for 5 hours using 1 U of Escherichia freundii endo S-galactosidase.
• the temperature at which KS is partially desulfated for 3 hours by the DMS method is preferably about 50 to 70 ° C, and more preferably 60 ° C. You. 37 ° C using 1U KSase II
• the temperature at which KS is partially desulfated by DMSO for 3 hours is approximately 65 ° C. It is easily understood by those skilled in the art that the above is preferable, 70 ° C or more is more preferable, and 80 ° C is extremely preferable.
• the constituent disaccharides of the reaction product (KS with adjusted sulfate group content) at temperatures of 40, 50, 60, 70, and 80 ° C when KS is partially desulfated by the DMS method for 3 hours
• the number of moles of sulfate groups per mole was 1.6, 1.4, 1.0, 0.8 and 0.3, respectively.
• the anthrone-positive fraction of the flow-through solution is concentrated, and cellulofine GC is 90sf (Cellulofine GC 90sf; Seikagaku Corporation)
• Gel filtration was carried out using a column (4 x 100 cm) manufactured by the company with water as a solvent.
• the eluate was detected by the Anthrone method, and the peaks corresponding to 6, 8, 10, and 12 sugars were collected, concentrated, desalted, and freeze-dried (yields were 253 mg for hexasaccharide, 324 mg for octasaccharide, 185 mg for decasaccharide and 54 for decasaccharide).
• 100 mg of 10 sugars is dissolved in 10 m of water, and applied to a 0DS column, Daisopack SP 120-25-25DS-B (manufactured by Daiso-Ichi Co., Ltd .; 2 x 50 cm) in 2 ml portions 5 times. Then, elution was performed using a concentration gradient system from water to a 20% methanol solution, and the decasaccharide fraction was concentrated and lyophilized to recover. The yield was 78 mg. Similarly, 85 mg and 82 mg of purified powder were recovered from 100 mg of each of the octasaccharide and hexasaccharide fractions.
• the concentration of thiobarbituric acid-positive fraction adsorbed on the Dowex column and eluted by a linear concentration gradient with 2 L of 5 mM to 200 mM NaCl solution is also similar to cellulofine GCL 90sf (Cellulofine GCL-90sf) Load onto the column, perform gel filtration with 0.2 NaCl solution, collect fractions corresponding to 9-saccharide, 11-saccharide, and 13-saccharide, purify each by repeated gel filtration on the same column, desalinate, freeze-dry did.
• sialyl N-acetyl lactosaminooligosaccharides of 9-, 11-, and 13-saccharides were added at 12 mg, 15 mg, and 15 mg, respectively. 7 m was obtained.
• the sialic acid, galactose and nitrogen contents were measured for the fractions of hexasaccharide, octasaccharide, decasaccharide, decasaccharide, decasaccharide, eleven sugar and thirteen sugar, respectively.
• the sialic acid content was measured by the thiobarbituric acid method
• the galactose content was measured by the anthrone method
• the nitrogen content was measured by elemental analysis. Table 2 shows the results.
• “Theoretical value” in Table 3 is the molecular weight determined from the following structural formula.
• Gal represents a galactose residue
• GlcNAc represents an N-acetylglycosamine residue
• NeuAc represents an N-acetyl neuraminic acid residue
• a glycosidic bond It has been confirmed that the molecular size (molecular weight) of the N-acetylsyllactosamine oligosaccharide obtained according to the present invention almost coincides with the theoretical value. Also, It was confirmed that N-acetyl lactosaminoligosaccharides having strangely different molecular sizes (molecular weight :) can be separated according to the size and size.
• a novel N-acetylsilactosamin oligosaccharide and a method for producing a novel N-acetylsilactosamin oligosaccharide and a method for producing a novel N-acetylsilactosamin oligosaccharide.
• the oligosaccharide of the present invention can be used as a material for synthesizing a ligand sugar chain of a selectin family, which is present only in trace amounts in nature.
• the ligand sugar chain of the selectin family can be used as a novel drug, especially as an anti-inflammatory agent.
• the oligosaccharide of the present invention can be produced on an industrial scale at low cost by the method 1 of the present invention.
• a sulfated N-acetylacetyltosamine oligosaccharide useful as an intermediate for producing the oligosaccharide of the present invention can be produced on an industrial scale at low cost.
• the sulfated N-acetyl lactosamine oligosaccharide may have a novel physiological activity, and is expected to be used as a pharmaceutical.

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